OUC-China - 2014.igem.org2014.igem.org/files/presentation/OUC-China_Championship.pdfMini plasmid OUC...
Transcript of OUC-China - 2014.igem.org2014.igem.org/files/presentation/OUC-China_Championship.pdfMini plasmid OUC...
OUC-China
lasmid dventurer
OUTLINE
ProjectOverview
Conjugation
Transfection
Model
Policy & Practice
1
Lentivirus: the long preparation
Lipofectamine: the toxicity
Particle bombardment: the high expense
Three Ways of Transfection
2
Overview
A novel model of plasmid transfer
Double plasmids system
Conjugation
Product the fusion protein TAT-H4
Lysis
Transfection3
Transfer exogenous geneticmaterial by conjugation
Reduce damage
Transfer efficiently
Zebrafish's intestines
Conjugation
4
Features a broad conjugation Spectrum
IncPα
The length of this plasmid is 60kb.
Carries many kinds of resistant genes
RP4 Conjugation Requirements Conjugation
5
Tra gene cluster
OriT nick site
1. Tra and Trb gene clusters 2.Conjugative transfer origin
Mating pair formation system(Mpf) RP4 relaxosome nick site
RP4 Conjugation Requirements Conjugation
6
Mini plasmid contains function gene and OriTsequence.
Plasmid RP4 create conjugative conditions.
Double-plasmid-system Conjugation
8
The double plasmids system
Design on Double-plasmid-system Conjugation
9
Resistances ofthe donor and recipient cell
HB101
Chloramphenicol(mini plasmid and plasmid RP4
provide)
TOP10
Streptomycin(genome provide)
ChlR
Result Conjugation
10
The experiment results
Result Conjugation
Str
Chl
Str & Chl
HB101 & Top10 Top10 HB101
Culture the donor, recipient, and mixture on selective medium 11
Electrophoretogram DNA Sequencing
Result Conjugation
3000bp
5000bp
1500bp
2000bp
750bp1000bp
500bp
250bp
100bp
This image is from Wikipedia
Mini plasmid OUC
12
Conjugation with Vibrio harveyi
Result Conjugation
control group experimental group
13
Result Conjugation
Str Chl Str & Chl
HB101 & Top10
Top10
HB101
OriT of another resistant plasmid
Culture the donor, recipient, and mixture on selective medium 14
Result Conjugation
15
Not finished yet.
But just a matter of time.
Produce TAT-H4 Lysis Transfection
Transfection
16
The function of TAT
Histone H4The structure of nucleosome
TAT-PTD
Design on Fusion Protein Transfection
17
Complete the Mini Plasmid Transfection
18
3000bp5000bp
1500bp2000bp
750bp1000bp
500bp
250bp100bp
TAT::H4 Transfection
The protein can bind plasmids
19
TAT:H4/DNAmass ratio
From Lane 1 to 6. TAT:H4/DNA mass ratio reached 8:1, 6:1, 4:1, 2:1, 1:1, 0:1. Lane 7 is pcDNA3.1(+)-EGFP and lane 8 is Marker DL5000
3000bp5000bp
1500bp2000bp
750bp1000bp
500bp
250bp
100bp
The protein can protect plasmids
TAT::H4 Transfection
20
TAT::H4 Transfection
The result of Rt-PCR
3000bp
1500bp
2000bp
750bp
1000bp
500bp
250bp
100bp
21
The efficiency of transfection of injecting TAT-H4-plasmid is higher 92.41% than injecting plasmid only at least.
TAT::H4 Transfection
22
OD:5mm2
Inducible promoter
Lysis
Design of Lysis Device Transfection
23
Design of Lysis Device Transfection
24
Design of Lysis Device Transfection
Concentration ofL-arabinose
Time after induction
3 hours 6 hours 9 hours
1 μM 0.322 0.745 0.901
3 μM 0.247 0.741 0.927
6 μM 0.209 0.733 0.891
10 μM 0.191 0.750 0.924
100 μM 0.183 0.721 0.848
10 mM 0.132 0.631 0.685
25
lysis
L-arabinose aTc Quorum
sensing
Analysis
of data
ODE
modelPromotion
Modeling
26
The device A has worked!
Using L-arabinose to Induce Lysis Modeling
The line chart of the experimental data
27
PJ23106
tetRPR0040
tetR
aTc
lysis
Mechanism
Using aTc to Induce Lysis Modeling
ODE
𝑑 𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑑𝑡
= 𝐾11
𝑎𝑇𝑐− 𝛼1 𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛 + 𝛼2
𝑑 𝑙𝑦𝑠𝑖𝑠
𝑑𝑡= 𝐾2
1
𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑛 + 𝛽 𝑎𝑇𝑐
28
Result Modeling
𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛 =𝐾1 + 𝛼1 𝑎𝑇𝑐 + 𝐶1𝑒
−𝛼2𝑡
𝛼2 𝑎𝑇𝑐
𝑙𝑦𝑠𝑖𝑠 = 𝐶2 +𝑡 𝐾2 + 𝛽 𝑎𝑇𝑐 𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛
𝑛
𝑡𝑒𝑡𝑅𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑛
29
The aTc additive amount
The amount of
lysed cells
Estimate
Control
Result Modeling
29
Input
Gene
Protein
LysisTopological structure
ODE
Application Modeling
𝑑 𝑝𝑟𝑜𝑡𝑒𝑖𝑛
𝑑𝑡= 𝐾1
𝑙
𝑖𝑛𝑝𝑢𝑡− 𝛼1 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 + 𝛼2
𝑑 𝑙𝑦𝑠𝑖𝑠
𝑑𝑡= 𝐾2
𝑙
𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑛 + 𝛽 𝑖𝑛𝑝𝑢𝑡
30
ODE
Self-lysis Device Modeling
Self-lysis Regulated by Quorum Sensing
𝑑 𝑐
𝑑𝑡=
𝛽3 𝑖𝑛𝑝𝑢𝑡 𝑛
𝐾1𝑛 + 𝑖𝑛𝑝𝑢𝑡 𝑛
− 𝛼3 𝑐
𝑑 𝑙
𝑑𝑡=
𝛽1𝐾𝑐1𝑚
𝐾𝑐1𝑚 + 𝑐 𝑚
+𝛽2 𝑙𝑢𝑥 𝑝
𝐾𝑐𝑝 + 𝑙𝑢𝑥 𝑝
− 𝛼1 𝑙
𝑑 𝑙𝑢𝑥
𝑑𝑡=
𝛽3𝑘𝑐2𝑞
𝑘𝑐2𝑞 + 𝑐 𝑞
− 𝛼2 𝑙𝑢𝑥
31
pH
pH=8 pH=5
Diagrams of [c] [lux] and [l]
Result Modeling
𝑚 = 4, 𝑛 = 4, 𝑝 = 4, 𝑞 = 4α1 = 2, α2 = 2, α3 = 2β1 = 2, β2 = 2, β3 = 2
𝑘1 = 1, 𝑘𝑐 = 1, 𝑘𝑐1 = 1, 𝑘𝑐2 = 1
32
Simplifying the RP4 plasmid
Self-lysis device automatically
Application in Oral DNA vaccine for fish
Future
33
Communication
Camp
Outreach
Policy & Practice
34
Qingdao Institute of Biomass Energy and Bioprocess Technology
The Central China iGEMers' Consortium
Beijing Normal University iGEM team Peking iGEM team
Communication Policy & Practice
35
The mini jamboree Outward bound
Ecology practice Ecology experiment
Camp & Class & Lecture Policy & Practice
The Fourth Science and Technology camp
36
Transport between cities Parts submission to iGEM officials
We have done…
Investigate the relevant government department and different express companies.
Write to the State Post Bureau of China.
Make a proposal to iGEM officials
Outreach Policy & Practice
The investigation of biological products transport
37
Outreach Policy & Practice
DNA vaccine
coated pellet
Fish feed
Use feed as the carrier of DNA vaccine
✓11 BioBricks to part registry
✓An innovative method to carry exogenous DNA from prokaryote to Eukaryote
✓Modeling standard for lysis device
✓ Policy and practice
Achievements
39
Name Type Description Designer Length
BBa_K1439000 Conjugation Origin of transfer for the RP4-plasmid nic region. Wenqi Li 350
BBa_K1439001 Composite This part contains a reporter gene BBa_J04450,
combined with OriTRP4. Used to test plasmid
mobility.
Wenqi Li 1427
BBa_K1439002 Composite This part contains a reporter gene BBa_J04450,
combined with OriTR. Used to test plasmid
mobility.
Wenqi Li 1448
BBa_K1439003 Composite [OriTR-RFP]+[lysis] Wenqi Li 3379
BBa_K1439004 Coding TAT-H4 Zhaoliang
Chen
393
BBa_K1439005 Coding TAT-PTD Zhaoliang
Chen
36
BBa_K1439006 Coding Histone H4 Zhaoliang
Chen
312
BBa_K1439007 Regulatory CMV promoter Zhaoliang
Chen
588
BBa_K1439008 Composite TAT-H4-B0015 Zhaoliang
Chen
530
BBa_K1439009 Device Lysis device induced by L-arabinose Ming Jiang 1923
BBa_K1439010 Device Lysis device induced by aTc Ming Jiang 2738
Biobricks
40
✓ Our new BioBrick Parts were designed and they can work as expected.
✓We characterized our including information and behavior.
✓We submitted our new part to the iGEM Parts Registry.
✓We sent two standard biological parts to Peking iGEM team. And we have helped Peking to test the two parts.
✓ During the work time, we did investigations about biological products transport and vaccines about our project.
Judging Form
41
Xiaohua Zhang Guanpin Yang
Acknowledgments
Instructors
42
Prof. Yunxiang MaoDr. Xianghong WangProf. Zhenmin BaoProf. Min Wang Prof. Chenguang LiuProf. Zhigang QiuDr. Xiaolong WangProf. Huarong Guo College of Marine Life Science,
Ocean University of China
Acknowledgments
43
END